The objective of this research is to elucidate the events and mechanisms that govern expression of fatty acid synthetase (FAS) genes and lipogenesis during differentiation of adipocytes or exposure of adipocytes to insulin or lipolytic hormones. 3T3-L1 preadipocytes are genetically homogeneous cells that differentiate into hormone responsive adipocytes in vitro. Regulation of FAS levels during differentiation of 3T3-L1 preadipocytes will be studied by (a) precisely measuring FAS specific activity by a sensitive radioisotope assay, (b) preparing anti-FAS serum and determining changes in mass of FAS by competitive displacement radioimmunoassays, (c) assaying the rates of FAS biosynthesis and degradation by an immunoprecipitation-SDS-polyacrylamide gel-autoradiography procedure, (d) quantitating relative FAS mRNA levels by in vitro translation and (e) purifying FAS mRNA and synthesizing a (3H) cDNA probe to directly determine FAS mRNA sequences and detect possible covalently-modified, nontranslatable FAS sequences. The same series of analyses will be performed on differentiated 3T3-L1 adipocytes after pre-treating cells with physiological and supraphysiological concentrations of insulin and lipolytic hormones. Finally, 3T3-L1 adipocytes will be studied as models for elucidating defects in the regulation of FAS concentration in insulin deficient and hyperinsulinemic states.